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Application of wnt/β-catenin signaling pathway inhibitor in the preparation of drugs for promoting cell apoptosis

A signaling pathway and inhibitor technology, applied in the field of molecular genetic biology, to achieve the effect of promoting apoptosis

Inactive Publication Date: 2011-12-07
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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However, this conclusion needs more experimental support

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  • Application of wnt/β-catenin signaling pathway inhibitor in the preparation of drugs for promoting cell apoptosis
  • Application of wnt/β-catenin signaling pathway inhibitor in the preparation of drugs for promoting cell apoptosis
  • Application of wnt/β-catenin signaling pathway inhibitor in the preparation of drugs for promoting cell apoptosis

Examples

Experimental program
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Embodiment 1

[0022] 1.1 Collection of porcine ovaries and selection of follicles

[0023] Fresh porcine ovaries were collected, placed in physiological saline containing gentamicin, transported to the laboratory at 37°C, and healthy ovaries were selected as experimental materials.

[0024] 1.2 Isolation and culture of granulosa cells

[0025] Before the experiment, put the cell culture medium and PBS in the water bath in the sterile cell room to preheat at 37°C, and put the equipment needed for the experiment into the cell for sterilization, culture bottles, high-pressure sterilized centrifuge tubes and 1mL Put the tip of the pipette into the ultra-clean workbench; wash the ovary twice with normal saline containing double antibodies, wash it with PBS for 3-4 times, and scrub it twice with alcohol cotton, and select follicles with a diameter of 2-5mm for granulosa cell collection; use Extract the follicular fluid and granulosa cells on the follicle wall with a syringe with a 20g needle, an...

Embodiment 2

[0050] Example 2 Effect of siRNA transfection of granulosa cells on the expression of β-catenin

[0051] 2.1 Cell transfection (taking 24-well plate as an example)

[0052] Refer to FuGENE HD Transfection Reagent Manual, using FuGENE Use the amount recommended in the HD Transfection Reagent instruction manual as a starting point, follow the steps below to determine the optimal interfering strand of siRNA and its interfering concentration.

[0053] Transfection method:

[0054] (1) When the cells reach a confluence of 40-80% under a microscope, replace the granulosa cells with an antibiotic-free and serum-free medium, and place them at 37°C, 5% CO 2 Attach to the wall for 24 hours in the incubator;

[0055] (2) A certain amount of diluted siRNA (20nM) was added dropwise to serum-free and antibiotic-free medium with a total volume of 51uL, which was solution A (amount of single well, calculated with final concentrations of 25, 50, 75 and 100nM respectively );

[0056] (3...

Embodiment 3

[0092] Example 3 Effect of siRNA transfection of granulosa cells on apoptosis and apoptosis-related factors

[0093] 3.1 Cell transfection: Same as Example 2, wherein the siRNA in the experimental group is only 75nM β-catenin-siRNA-365.

[0094] 3.2 Annexin V / PI double staining flow cytometry to measure the apoptosis rate of granulosa cells

[0095] (1) 36h after β-catenin-siRNA-365 transfection, the cells were digested with EDTA-free trypsin;

[0096] (2) Collect suspended cells by centrifugation at 2000rpm for 5min;

[0097] (3) Wash the cells twice with PBS and centrifuge at 2000rpm for 5min;

[0098] (4) Add 500uL Binding Buffer to suspend the cells;

[0099] (6) After adding 5ul Annexin V-FITC and mixing, add Propidium Iodide and mix;

[0100] (7) React at room temperature and avoid light for 5-15 minutes;

[0101] (8) Perform flow cytometry detection within 1 hour, excitation wavelength Ex=488nm, emission wavelength Em=530nm, the results are shown in Table 3, Figu...

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Abstract

The invention belongs to the field of molecular genetics biology and discloses application of a Wnt / beta-catenin signal channel inhibitor to the preparation of a medicament for promoting apoptosis. The Wnt / beta-catenin signal channel inhibitor is preferably siRNA (small interfering Ribonucleic Acid) of beta-catenin. Chemical synthesis beta-catenin-siRNA is designed specific to beta-catenin genes,and granular cells are transferred with a lipoplast, so that the beta-catenin genes in pig ovary granular cells are decreased. A result shows that apoptotic cells in an experimental group are remarkably increased from 8.80 percent to 15.85 percent in comparison to a blank group after beta-catenin expression is inhibited, the difference is remarkable, and the beta-catenin-siRNA can promote the apoptosis of granular cells.

Description

technical field [0001] The invention belongs to the field of molecular genetic biology, and relates to the application of Wnt / β-catenin signaling pathway inhibitors in the preparation of drugs for promoting cell apoptosis. Background technique [0002] β-catenin (β-catenin), as the core transcription factor in the Wnt signaling pathway, plays an important role in the process of ovarian development. Or cells have important effects. Knockout of Wnt4 can lead to partial sex reversal and oocyte failure in mice before birth, and follicle-like structures containing disordered and pleomorphic granulosa cells are formed in granulosa cells of β-catenin knockout mice, Can lead to the occurrence of granulosa cell carcinoma. The above findings indicate that Wnt / β-catenin has a profound impact on the proliferation, differentiation and survival of granulosa cells, and thus plays an important role in maintaining the normal function of the ovary. However, this conclusion still needs more...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K48/00A61P35/00
Inventor 徐银学王莉张宝乐侯艳君
Owner NANJING AGRICULTURAL UNIVERSITY
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